J Mater Sci: Mater Med (2015) 26:17

DOI 10.1007/s10856-014-5333-y

BIOMATERIALS SYNTHESIS AND CHARACTERIZATION

Structural and biological evaluation of lignin addition to simple

and silver-doped hydroxyapatite thin films synthesized
by matrix-assisted pulsed laser evaporation
A. Jankovic S. Erakovic C. Ristoscu N. Mihailescu (Serban)
L. Duta A. Visan G. E. Stan A. C. Popa M. A. Husanu C. R. Luculescu
V. V. Srdic Dj. Janackovic V. Miskovic-Stankovic C. Bleotu
M. C. Chifiriuc I. N. Mihailescu

Received: 27 February 2014 / Accepted: 28 July 2014

Springer Science+Business Media New York 2014

Abstract We report on thin film deposition by matrixassisted pulsed laser evaporation of simple hydroxyapatite
(HA) or silver (Ag) doped HA combined with the natural
biopolymer organosolv lignin (Lig) (Ag:HALig). Solid
cryogenic target of aqueous dispersions of Ag:HALig
composite and its counterpart without silver (HALig)
were prepared for evaporation using a KrF* excimer laser
source. The expulsed material was assembled onto TiO2/Ti
substrata or silicon wafers and subjected to physical
chemical investigations. Smooth, uniform films adherent to
substratum were observed. The chemical analyses confirmed the presence of the HA components, but also evidenced traces of Ag and Lig. Deposited HA was Ca
deficient, which is indicative of a film with increased solubility. Recorded X-ray Diffraction patterns were characteristic for amorphous films. Lig presence in thin films was
undoubtedly proved by both X-ray Photoelectron and
Fourier Transform Infra-Red Spectroscopy analyses. The
microbiological evaluation showed that the newly assembled surfaces exhibited an inhibitory activity both on the

initial steps of biofilm forming, and on mature bacterial

and fungal biofilm development. The intensity of the antibiofilm activity was positively influenced by the presence
of the Lig and/or Ag, in the case of Staphylococcus aureus,
Pseudomonas aeruginosa and Candida famata biofilms.
The obtained surfaces exhibited a low cytotoxicity toward
human mesenchymal stem cells, being therefore promising
candidates for fabricating implantable biomaterials with
increased biocompatibility and resistance to microbial
colonization and further biofilm development.

A. Jankovic
S. Erakovic
Innovation Center, Faculty of Technology and Metallurgy,
University of Belgrade, 11000 Belgrade, Serbia

V. V. Srdic
Department of Materials Engineering Faculty of Technology,
University of Novi Sad, 21000 Novi Sad, Serbia

C. Ristoscu
N. Mihailescu (Serban)
L. Duta
A. Visan

C. R. Luculescu
I. N. Mihailescu (&)
National Institute for Lasers, Plasma and Radiation Physics,
077125 Magurele, Ilfov, Romania
e-mail: ion.mihailescu@inflpr.ro

Dj. Janackovic
V. Miskovic-Stankovic

Faculty of Technology and Metallurgy, University of Belgrade,
11000 Belgrade, Serbia

G. E. Stan
A. C. Popa
M. A. Husanu

National Institute of Materials Physics, 077125 Magurele, Ilfov,
Romania
A. C. Popa
Army Centre for Medical Research, 020012 Bucharest, Romania

1 Introduction
Titanium (Ti) is widely used as an implantable biomaterial
for medical devices like dental implants, fracture fixations
and joint replacements [1, 2], due to its high strength,
toughness, and durability. However, Ti requires an appropriate surface biofunctionalization to increase hard and/or
soft tissue compatibility and to exhibit antimicrobial

C. Bleotu
Stefan S. Nicolau Institute of Virology, 030304 Bucharest,
Romania
M. C. Chifiriuc
Department of Microbiology, Faculty of Biology, University of
Bucharest, 060101 Bucharest, Romania

123

17

Page 2 of 14

properties for the inhibition of biofilm formation. Biofilm

represents a microbial community that irreversibly attaches
to a host surface, being protected by a self-secreted
extracellular polymeric matrix and other complex mechanisms from the action of antibiotics, disinfectants and host
immune effectors.
Despite their frequent presence as commensal bacteria
on the human skin and mucous surfaces, staphylococci are
the most frequent causes of biofilm-associated infections,
especially in intensive care patients. Studies have shown
that Staphylococcus aureus could form static biofilms on
different modified Ti surfaces. The Ti coating with sodium
hyaluronate thin films [3], the hydrophobic and superhydrophobic surfaces resulting from Ti surface modification
by TiO2 nanotube arrays or pure Ti treated with 1H, 1H,
2H, 2H-perfluorooctyl-triethoxysilane [4], as well as Ag
nanoparticle-modified Ti [2] proved to be efficient in preventing S. aureus biofilm-associated infections.
Pseudomonas aeruginosa is an opportunistic human
pathogen causing a wide range of infections, from wound
infections to lung diseases in patients with cystic fibrosis
and medical device-associated infections [5]. Studies have
shown that the solgel TiO2 with Ag or Degussa TiO2 with
Ag attenuate P. aeruginosa adherence and growth [6].
Fungi most commonly related to biofilm-associated
infections belong to the genus Candida, being usually
connected with indwelling medical devices. The development and characterization of C. albicans biofilms formed
on bioprosthetic materials have been intensively investigated [7]. Malm et al. [8] studied C. famata biofilm
development on glass slides by microscopic examination. It
was observed that after 24 h of incubation, C. famata
biofilm is still growing, and after 72 h it reaches the stage
of a mature biofilm, accompanied by changes in morphological reorganization.
Although Ti does not possess intrinsic antifungal properties, TiO2 can act as a photocatalyst for the decomposition of organic compounds under UV irradiation, reducing
the viability of C. albicans [9, 10]. Ti-coated silicone was
thought to prevent Candida biofilm formation on voice
prostheses by increasing the smoothness of the material
surface [11].
Due to the dramatic increase of traumatic, pathological or
surgical injury cases that require hard-tissue implants, many
techniques for Ti surface modification, such as coating with
hydroxyapatite [HA, Ca10(PO4)6(OH)2], have been developed, some of them being already commercialized [12, 13].
HA is an excellent biocompatible material, and it is largely
used nowadays in medicine in all forms (bulk, coating,
emulsion) [14], due to its chemical and structural similarities
to the mineral part of the human bone tissue.
Doping HA with antimicrobial agents, especially with
silver (Ag) ions, is a promising approach to address the

123

J Mater Sci: Mater Med (2015) 26:17

difficult problem of microbial biofilm-associated infections

on medical prostheses [15]. As biofilms are the most
common mode of bacterial growth and lead consistently to
clinical infections (up to 80 %), especially because of their
high antibiotic resistance, there is a stringent need for new
and efficient implant functionalization solutions. Biofilms
on indwelling medical devices result in significant morbidity and mortality and have a substantial impact on
healthcare systems worldwide [16]. Biomaterial-associated
infection incidence is increasing proportionally to the
number of people gaining access to medical device technologies worldwide, but also due to the emerging microbial
resistance to current antibiotics [17]. The clinical experience showed that Ti devices are frequently colonized by
microbial strains and the subsequent biofilm formation
represents a huge complication in implant surgery [18].
Recent research advances in understanding the interaction
between microbial biofilms and Ti surface contributed to the
development of novel preventive strategies to control medical device-related infections. The focus is on obtaining
improved biomaterials with increased resistance to microbial
colonization via surface physicalchemical modifications.
Today, Ag and Ag nanoparticles are mostly used in a wide
range of applications: healthcare, textile, food, hard surface
material industry or domiciliary applications [18, 19]. Ag
ions possess strong antimicrobial properties, which, correlated with no immediate and serious risk for human health,
have led to an extensive use of Ag-based products in many
biomedical applications [18, 19].
Lignin (Lig) is a complex, amorphous organic polymer
found in plant tissues, usually bound to cellulose. Phenylpropane units in Lig are cross-linked to each other by various
chemical bonds. Lig is a well-known and important source of
natural antimicrobial and antifungal compounds [20].
We have previously reported on composite HALig
materials that could be used as biomedical coatings [21,
22] exhibiting enhanced bioactivity and osteoconductivity.
The novel concept of engaging natural biopolymer Lig in
composite coatings was studiedso far by electrophoretic
deposition only [23, 24]. However, an unaltered incorporation of this specific organic material could provide a
composite with enhanced stability and improved interconnected structure, which will increase the coating cohesion.
Matrix-assisted pulsed laser evaporation (MAPLE)
technique is derived from Pulsed Laser Deposition (PLD),
developed for controlled assembly of biopolymer thin films
[25, 26]. The method is based upon a cryogenic approach
which secures the transfer of organic macromolecular
materials. The key components are dissolved or suspended
in an appropriate volatile solvent, which is flash-frozen to
form a target. Due to the low concentration of biomolecules, the laser photons mostly react with the frozen
matrix (solvent), which vaporizes and gets pumped out of

J Mater Sci: Mater Med (2015) 26:17

the deposition chamber. Thus, organic molecules of the

frozen target can get transferred undamaged and not
degraded to the substratum [2729].
In a recent paper, we have demonstrated the positive
role of the Ti surface modification by arrays of TiO2
nanotubes on the biocompatible and antifungal response of
top HA layers deposited by PLD [30]. In the present
research, we aimed to obtain biomimetic ceramic-polymer
composite coatings for medical Ti implants modified with
100 nm diameter TiO2 nanotubes (fabricated by anodization of Ti plates) by employing MAPLE as fabrication
method. Composite HALig and Ag doped HALig were
transferred by this technique, their structure and composition were assessed, as well as their cytotoxicity against
human Whartons Jelly-derived mesenchymal stromal cells
(WJ-MSCs) and antimicrobial efficiency against Grampositive (S. aureus ATCC 6533), Gram-negative (P.
aeruginosa ATCC 27853) bacterial and fungal (C. famata
30) strains.
The main purpose of the microbiological assay was to
evaluate the anti-biofilm efficiency of thin films of simple
hydroxyapatite (HA) or silver (Ag) doped HA combined
with the natural biopolymer organosolv (Lig) (Ag:HA
Lig), deposited by MAPLE.

Page 3 of 14

17

order to obtain HA or Ag/HA powder, Ca9.95Ag0.05

(PO4)6(OH)2. After the required quantity of phosphoric
acid was introduced, the pH reached a value of 7.47.6.
The obtained suspension was preheated to (94 1) C for
30 min and stirred for another 30 min. After sedimentation,
the upper clear solution layer was decanted from precipitate. The suspension was then spray-dried at (120 5) C
into granulated powder.
2.3 Cryogenic target preparation and mounting
Organosolv Lig (extracted by the Alcell process) was used
for preparing organicinorganic composite targets.
Solutions consisting of HA or Ag:HA powders (10 %
w/v) and Lig (1 % w/v) dissolved in distilled water were
homogenized by rapid vortexing and flash frozen in a liquid
nitrogen cooled copper container. The container with the
obtained frozen target (HALig or Ag:HALig pastilles) was
then mounted on a cryogenic holder inside the deposition
chamber and rotated at 10 rpm to avoid local overheating and
excess ablation during multipulse laser irradiation. The
holder was submerged in liquid nitrogen flow in order to keep
the targets frozen during the experiments.
2.4 MAPLE experiment

2 Experimental
2.1 Preparation of Ti substrata
Pure Ti foils (20 9 10 9 0.25) mm3 in size and 99.7 %
purity (Sigma Aldrich) were used as substrata for the
growth of arrays of TiO2 nanotubes by anodization technique described in Ref. 30. As prepared, TiO2/Ti substrata
were degreased in acetone, then in ethanol, each for 30 min
in ultrasonic bath and finally kept in ethanol until deposition. Just before their introduction into the reaction
chamber, the substrata were rinsed with deionized water
and jet-dried with N2.
2.2 Preparation of HA and Ag/HA powders
For the preparation of HA powder with and without Ag we
used a modified chemical precipitation method [21]. Calcium oxide, synthesized by aerobic calcination of CaCO3
for 5 h at 1000 C, was placed in a reaction vessel with Ag
nitrate (AgNO3), in the case of Ag:HA, and phosphoric
acid. The reaction was conducted in a step-wise manner. A
stoichiometric amount of the resulting calcium oxide was
mixed and stirred in distilled water for 10 min. Afterwards,
AgNO3 solution was added to the suspension, to reach a
final concentration of Ag ion of (0.6 0.1) wt%. Finally,
phosphoric acid was added drop-wise to the suspension in

HALig and Ag:HALig composite coatings deposition

was performed at room temperature in a pressure of 6.5 Pa
onto TiO2/Ti substrata. The target-to-substratum separation
distance was 35 mm. A pulsed KrF* excimer laser source
(k = 248 nm, sFWHM B 25 ns) operating at 10 Hz was
used for target evaporation. The laser beam was incident
onto the target surface at 45. The spot size was of 25 mm2.
A total of 35,000 pulses with an incident laser fluence of
0.7 Jcm-2 were applied for the deposition of each structure. It is important to note that this fluence level is about
five times lower than in conventional PLD of inorganic
materials, as a supplementary precaution adopted in
MAPLE to protect Lig molecules against extensive laser
beam irradiation. Twin samples were deposited on \111[
single-crystalline Si wafers.
2.5 Morphological, structural and compositional
characterization
The surface morphology of the deposited films was
investigated by scanning electron microscopy (SEM) with
a Carl Zeiss EVO 50 XVP instrument, operated at 30 kV
acceleration voltage and 10 lA beam current, under secondary electron mode. No conductive coating was applied
onto film surface. Cross-sectional SEM images were
recorded on HALig and Ag:HALig films deposited on Si
wafers in order to evaluate their thickness.

123

17

Page 4 of 14

Composition analysis was performed by energy dispersive spectroscopy (EDS), with a SiLi EDAX Inc. detector,
operated at 20 kV. The measurements were conducted
in duplicate, on different, relatively large regions of
(250 9 250) lm2.
The crystalline status of the MAPLE thin films was
evaluated by Grazing Incidence X-ray diffraction (GIXRD)
using a Bruker D8 Advance diffractometer, in parallel
beam setting, equipped with a Cu target X-ray tube. The
incidence angle was set at 2, and the scattered intensity
was scanned in the range 2050 (2h), with a step size of
0.04, and 50 s per step.
X-ray photoelectron spectroscopy (XPS) analysis was
performed to assess the Lig transfer. The XPS measurements were conducted in a SPECS dedicated surface science facility, keeping the base pressure during
measurements below 10-8 Pa. The spectra were recorded
using the Al Ka1 monochromatized radiation (E =
1486.74 eV) in an analysis chamber equipped with a
150 mm hemispherical electron energy analyzer (Phoibos).
Fixed analyzer transmission mode was operated with pass
energy of 20 eV and step energy of 0.05 eV. The estimated
combined (source ? analyzer) resolution was about
(0.75 0.025) eV. During the XPS measurements, a flood
gun operating at 1 eV acceleration energy and 100 lA
electron current was used in order to achieve sample
neutralization.
The short-range order analysis and the detection of the
functional groups present in the MAPLE films was carried
out by Fourier transform infra-red (FTIR) spectroscopy in
attenuated total reflection (ATR) mode using a Perkin
Elmer BX Spectrum-Pike spectrometer equipped with a
Pike-MIRacle ATR diamond head of 1.8 mm in diameter.
The spectra were collected over a range of (4000550)
cm-1 by recording 150 individual scans at 4 cm-1 resolution. During acquisition, the spectrometer chamber was
continuously purged with nitrogen to maintain a dry
environment.
2.6 Biological assays
2.6.1 Cytotoxicity assay
The biological compatibility of the MAPLE composite
coatings was assessed by cultivating human Whartons
Jelly-derived Mesenchymal Stromal Cells (WJ-MSCs) on
their surface. Quantification of cells was performed using
propidium iodide (PI). To this purpose, the obtained
specimens were sterilized by UV irradiation and placed in
35 mm diameter Petri dishes. In each Petri dish 3 9 105
mesenchymal cells were added. The monolayer morphology was evaluated after 24 h, by fixing the cells with 70 %
alcohol and staining the monolayer with 5 lg/mL PI. The

123

J Mater Sci: Mater Med (2015) 26:17

stained specimens have been examined and photographed

in fluorescent microscopy [31].
2.6.2 Microbial biofilm assay
The microbial adherence ability and biofilm development
on the functionalized surfaces have been investigated by
two culture-based methods, using Gram-positive (S. aureus
ATCC 6533), Gram-negative (P. aeruginosa ATCC
27853) bacterial and fungal (C. famata 30) strains. The
specimens (composite coatings and pure HA controls) of
the same size have been distributed in the multi-well
plastic plates, and exposed to UV sterilization for 30 min.
The pure HA controls have been synthesized by PLD in
optimized conditions [30]. All experiments were performed
in duplicate. Thereafter, the liquid culture medium (nutrient broth) was added over the slide specimens. Each well
was inoculated with a microbial inoculum with a density
corresponding to 0.5 MacFarland density prepared in
sterile saline. Each microbial strain was inoculated in two
wells containing the same specimen. Thus prepared samples were incubated at 37 C, in order to allow microbial
strains to multiply and adhere to the deposited Ti plates,
distributed in each well.
After 24, 48 and 72 h, respectively, the specimens were
extracted, washed three times in sterile saline, in order to
remove the non-adherent bacteria and moved in sterile
plastic wells. Fresh culture medium was thereafter added
and the multi-well plates were further incubated at 37 C
for 24 h. This point forward, the specimens have been
treated differently, in order to assess:
a)

b)

The total biofilm (viable and dead) cells

For the purpose of our assay, after incubation, the
density of the obtained cultures recovered after the
multiplication of microbial cells adhered to the tested
substrata was measured at 600 nm.
The viable cells embedded into the biofilms developed on different specimens.
Ten-fold dilutions were prepared from the cultures
recovered after the multiplication of microbial cells
adhered to the tested substrata in order to count the
Colony Forming Units (CFU) and to assess the viable
cell counts (VCCs) of the respective cultures. For this
purpose, the adhered cells have been removed from
samples by vortexing and brief sonication. Serial
dilutions ranging from 10-1 to 10-30 of the obtained
inocula have been spotted on Muller-Hinton agar,
incubated for 24 h at 37 C and assessed for VCCs.
An amount of 5 ll of the chosen dilution was spotted
in duplicates on the solid medium. The resulting
colonies have been numbered and the average value
was submitted to dilution and volume correction. The

J Mater Sci: Mater Med (2015) 26:17

final value was expressed in CFU/mL. Performing the

assay in this manner, we were able to assess the
influence of different tested substrata on the adherence and the dynamics of microbial biofilm development by selected microbial strains.

3 Results and discussion

Page 5 of 14

17

of *1.33 (inferior to 1.67 theoretical Ca/P ratio, characteristic to stoichiometric HA). Such a deviation could be
associated with Ca/P ratio dependence on substratum
temperature and laser incident fluence [25, 28, 30]. Small
traces of Ag (*0.57 wt%) have been detected in the case
of Ag:HALig films. However, because of the low accuracy of the EDS technique, this value should be considered
as a rough approximation only.

3.1 SEMEDS

3.2 XRD

Typical top-view SEM images of the HALig and Ag:HA

Lig films are displayed in Fig. 1. MAPLE deposition resulted
in rather smooth films with a homogenous and pore-free
microstructure, without particular morphological features.
Rough surface of the films was reported starting from the
same nanohydroxyapatite powder composite with Lig, when
using the electrophoretic deposition [21]. As an important
note, no remarkable morphological differences have been
evidenced between the HALig and Ag:HALig films.
The cross-sectional SEM images collected in case of
films deposited onto Si wafers, revealed the compact look
of these MAPLE coatings with good adhesion to the substratum. A thickness of *180 10 nm has been estimated
based upon the cross-sectional SEM analyses. A typical
cross-SEM image of the Ag:HALig film grown on a silicon wafer is visible in Fig. 1b-inset.
The qualitative EDS analyses (data not shown) revealed
the high purity of films and indicated the presence of all
elements of HA, along with carbon content for both types
of films, suggesting the incorporation of Lig. Small traces
of Ag have been detected in the case of Ag:HALig films.
The quantitative EDS estimation indicated the synthesis of
a calcium deficient HA phase, as the atomic Ca/P ratio was
slightly altered during the ablation process down to a value

The GIXRD patterns revealed the Ti substratum maxima

only, suggesting that the TiO2 nanotubes, as well as the
HALig and the Ag:HALig coatings synthesized by
MAPLE were amorphous within the experimental sensitivity limit of the apparatus. A typical GIXRD pattern (for
the Ag:HA/TiO2/Ti film) is presented in Fig. 2. For comparison, the reference files of hydroxyapatite (ICDD:
00-009-0432), anatase (ICDD: 00-021-1272) and rutile
((ICDD: 00-021-1276) are superimposed on the graph.

Fig. 1 Top-view SEM micrographs of the HALig (a) and Ag:HA

Lig (b) films deposited onto TiO2/Ti substrata by MAPLE. Inset:
cross-view SEM micrograph of Ag:HALig film deposited onto
silicon wafer

3.3 XPS
The XPS spectra were recorded for the pure HA, HALig
and Ag:HALig films.
As known, charging effects may arise during measurements, resulting in an apparent shift of core-level XPS lines
[32]. Mitigating this effect usually involves using a floodgun corroborated to overall shift of all lines with values
that correlate to C1 s line at 284.5 eV, considered as
standard. In our case such calibration is difficult since C1 s
band has a complex structure, featured by several components. In fact, their assignment is essential in identifying
the Lig signature in the XPS spectrum.

Fig. 2 Typical GIXRD pattern of a Ag:HALig/TiO2/Ti film deposited by MAPLE

123

17

Page 6 of 14

J Mater Sci: Mater Med (2015) 26:17

In order to identify the contamination-associated component, a 30 s Ar? sputtering was performed at accelerating voltage of 3 kV resulting in an ion current of
*15 9 10-6 A. Assuming that every incoming ion pullsout a surface atom, one estimates that in 30 s we remove
2 nm approximately from the surface layer. Based on this
assumption, we expect that the signature provided by C1 s
spectrum entirely belongs to pure HA, HALig and
Ag:HALig composite films, free of any contamination.
Consequently, C-bonded carbon line was identified as the
component with the most evident intensity drop-off, and it
was kept at 284.5 eV and used as calibration line.
The presence of Lig was demonstrated in both HALig
and in the more complicated case of Ag:HALig. For
discussion, we therefore focused on a comparison of the
XPS data between pure HA and Ag:HALig coatings. The
intensity variation of the C1 s XPS components after the
sputtering cycle is given for the two cases (Fig. 3). The Lig
signature was revealed to be dispersed in the HA matrix, as
evidenced by a massive increase of the C-bonded carbon

signature, accompanied by a slight increase of the component associated with oxygen-bonded C or oxygen-containing radicals (Table 1).
The indisputable proof that the Lig has been effectively
transferred into the HA composite film would consist in
determining the experimental stoichiometry fraction xC:yO
from the XPS data considering the addition of 10 % Lig
into the HA matrix.
From the experimental stoichiometry inferred for the
pure HA case aC:bO and that of the HALig composite
0.9[aC:bO] ? 0.1[xC:yO], the values obtained were
x = 11, y = 4.4. These values closely correspond to the
Lig theoretic stoichiometry of the three monolignols
(C9H10O2/C10H12O3/C11H14O4) (Fig. 4) which lead to Lig
formation by polymerization. As a crosscheck and as
suggested in previous studies [3335], we have also calculated the theoretical stoichiometry of the dopant using
the integral amplitudes of the C1 s and O1 s peaks. Comparable results have been obtained, i.e. x = 11, y = 3.75.
These findings validate the experimental results.

Fig. 3 C 1 s core level high resolution XPS spectra of pure HA (a) and Ag:HALig (b) films
Table 1 C 1 s XPS core level sub-components areas
% C for as-introduced samples

% C for 30 s sputtered samples

CC/CH

COH/COR

C = O/HOCOR

C=O

CC/CH

COH/COR

C = O/HOCOR

C=O

Pure HA

41.86

41.79

9.70

6.65

28.33

57.37

10.35

3.95

Ag:HALig

76.7

17.08

1.91

4.31

70.42

27.10

2.48

123

J Mater Sci: Mater Med (2015) 26:17

Fig. 4 Theoretical stoichiometry and chemical formulas of the three

monolignols: (a) hydroxyphenyl (C9H10O2), (b) guaiacyl (C10H12O3)
and (c) syringyl (C11H14O4) [36]

3.4 ATR-FTIR
FTIR spectroscopy was applied for identifying the functional groups and the degree of short-range ordering in the
deposited films. Emphasis was put on identifying the distinct chemical bonds of Lig and the degree of macromolecule decomposition during MAPLE transfer, if any.
The ATR-FTIR spectra of the original Lig powder, pure
HA powder, and pure HA and Ag:HALig films (deposited
under optimized conditions) are shown comparatively in
Fig. 5. The assignment for the IR vibration bands is given
in Table 2.
In the fingerprint region (1800550 cm-1), Lig powder
exhibits an intricate spectrum with numerous sharp and discrete absorption bands due to its main molecular components
(Table 2). HALig and Ag:HALig coatings had similar
envelopes dominated by the typical vibration bands of HA
[37]: the m4 symmetric bending, m1 symmetric and m3 asymmetric stretching modes of phosphate groups, along with the
libration mode of structural OH (see Table 2). Because of the
complex composition of Ag:HALig coatings, we chose to
present in Fig. 5e the IR spectrum of this film only.
In the (1200550) cm-1 wave number region the
prominent HA bands are superimposed to some of the Lig
bands, partly obscuring them. Moreover, in the (890660)
cm-1 region, the intense band of the TiO and TiOTi
skeletal vibrations is brought in by the underlayer of TiO2
nanotubes [38]. However, the Lig contribution can be
hinted by a more complex shape of the IR envelope in the
case of Ag:HALig composite film (Fig. 5e) with respect
to the pure HA film and powder spectra (Fig. 5c, d).

Page 7 of 14

17

The undeniable evidence of the Lig macromolecule

transfer is revealed by the distinct bands of Lig visible in
the (18001200) cm-1 (Fig. 5a, e, inset) and the
(31001200) cm-1 (Fig. 5b, f) wave number regions [39
41]. The presence, in these specific spectral regions, of all
Lig vibration bands, suggests that the Lig material is not
altered during the MAPLE transfer, the slight shifts being
induced rather by the molecular interactions with the HA
matrix than by its degradation.
The broader IR spectrum in the case of Ag:HALig
composite film also indicates that a short-range ordering
alteration occurred as a consequence of intermolecular
interactions between the HA film matrix and the Lig
components. Differences in the absorbance values and
shape of the bands were also detected in the infrared
spectra, pleading as well for intimate structural modification induced by the Lig embedment in the HA film matrix.
The stretching modes of guaiacyl (G) and syringyl (S),
archetypal for Lig, were evidenced in the composite films
at the 1205 (G), 1283 (G), and 1320 cm-1 (S), respectively. The guaiacyl IR bands are dominant which suggests
that a larger amount of G units is present in the film. Higher
G/S ratios could increase cross-linking of the Lig molecules [40] dispersed in the entire HA matrix, and thus,
could contribute to the augmentation of the mechanical
properties of the film and a more durable composite
material.
3.5 Effect of the prepared samples on WJ-MSCs
viability
Stem cells and progenitor cells are promising candidates
for the development of efficient therapeutic and regenerative strategies, with a large spectrum of clinical applications, including biomaterials and tissue engineering [45].
MSCs are adult stem cells able to differentiate into a
variety of cell types in vitro, but also to engraft in vivo [46,
47]. We therefore decided to use MSC for our in vitro
cytotoxicity tests, with the conviction that the results would
be a solid foundation for future in vivo biocompatibility
studies of the MAPLE coatings.
We would like to emphasize that no significant changes
were observed in the morphology of WJ-MSCs, when
grown on the surface of the tested materials. The experiments have shown that the MAPLE composite coatings
exhibited no toxicity towards human cells and allowed a
sustained growth of WJ-MSCs.
It appears that the presence of Lig improves the biocompatibility of the HA coated TiO2/Ti, by promoting the
growth of adhered cells, clearly supporting the suitability
of our composites for developing future biomaterials with
increased biocompatibility (Fig. 6).

123

17

Page 8 of 14

J Mater Sci: Mater Med (2015) 26:17

Fig. 5 ATR-FTIR spectra of

Lig powder (a, b), pure HA film
(c), pure HA powder (SigmaAldrich) (d) and Ag:HALig
film (e, f) in the spectral
regions: 1800550 cm-1 (a, c,
d, e) and 31002700 cm-1 (b, f)

3.6 Microbial assay results

Our initial hypothesis was that adding Lig to HA-films
doped with Ag ions would yield a material with improved
antimicrobial properties. To test this hypothesis, we have
examined the anti-biofilm efficiency of the bioactive
composite coatings, by using two well-established microbiological assays. One is based upon the assessment of
VCCs, whilst the second resorts to bacterial culture density
measurements, for the quantification of total microbial
biofilm developed on the materials obtained at different
time intervals, i.e. 24, 48 and 72 h, respectively. These two
approaches could provide complementary information

123

regarding the number of viable cells embedded in the

biofilm (viable cell counts) as well as the density of
microbial biofilm (comprised of both viable or dead cells and the biofilm matrix).
The temporal dynamics of biofilms formed by microbial
species, either fungi or bacteria are different, as it is their
resistance to various antimicrobial agents [48, 49]. However, research performed in many biofilm-forming organisms has revealed that the development of a biofilm is a
two-step process involving an initial attachment and a
subsequent maturation phase, which are physiologically
different from each other and require phase-specific factors. A final dispersal phase involves the detachment of

J Mater Sci: Mater Med (2015) 26:17

Page 9 of 14

17

Table 2 Assignment of ATR-FTIR vibration bands for the lignin powder, pure synthetic HA powder (Sigma-Aldrich), pure HA film, and
Ag:HALig composite film
Observed IR bands (cm-1)

Bands assignment

Pure Lig

Pure HA powder

Pure HA film

Ag:HALig film

568

572

604

600

604

638

630

632

628

Librational mode of (OH)- groups [37]

COH out-of-plane bending [42]

731

CH bonds on the benzene rings [43]

Asymmetric bending (m4) of (PO4)3- groups [37]

Asymmetric bending (m4) of (PO4)3- groups [37]

756

749

Asymmetric bending of HCCH groups [39]

809

TiO vibrations [38]

827

CH out-of-plane in position 2 and 6 of syringyl (S) and in all

positions guaiacyl (G) units [39, 40]

871

Vibrations of (HPO4)2- ions [37]

884

CH deformation vibration of cellulose [39]

913

911

CH bending of S units [39]

961

961

942

Symmetric stretching (m1) of (PO4)3- groups [37]

969

=CH out-of-plane deformation [39, 41]

1029

1021

1040

1043

CO stretching of cellulose [39, 41]

Asymmetric stretching (m3) of (PO4)3- groups [37]

1086

1093

1089

Asymmetric stretching (m3) of (PO4)3- groups [37]

1111

C=O stretching [41]

1143

1146

Vibrations of (HPO4)2- ions [37]

1154

1168

COC asymetric stretching in cellulose [39, 41]

1211

1205

CC, CO and C=O stretching of G units [39, 40]

1270

1283

CO stretching of G units [39, 41]

1327

1320

CO stretching of S units [39, 41]

1366

1345

CH symmetric bending in cellulose [40, 41]

1424

1422

CH in plane bending in Lig [39, 41]

1457

1463

CH bending of methyl and methylene groups [39, 41]

1514

1512

C = C stretching of the aromatic ring (G) [40, 41]

1596

1573

C=C stretching of the aromatic ring (S) [41]

1667

1655

C=O stretching in conjugated p-subst. aryl ketones [40]

1702

1698

C=O stretching in unconjugated aldehyde, ketone, carbonyls

or ester groups [39, 40]

2740

2763

Aldehyde CH stretch [39, 41]

2843

2856

Asymmetric CH stretching in aromatic methoxyl groups and

in methyl and methylene groups of side chains [41]

2937

2921

2968

2972

Symmetric CH stretching in aromatic methoxyl groups and in

methyl and methylene groups of side chains [41]
sp3 hybridized CH [44]

3006

sp2 hybridized CH [44]

single cells or cell clusters promoting the bacterial dissemination [50]. In the maturation phase, bacterial cells
proliferate and produce an extracellular matrix consisting
of several secreted polymers, such as exopolysaccharides,
teichoic acids and specific proteins, as well as DNA originating from the lysed bacteria [51].
The observed dynamics of S. aureus biofilm formation
varied depending on the tested specimen.

The biofilm developed on the pure HA coating control

specimen showed a growth peak at 48 h, the number of
viable bacterial cells recovered at 48 h remaining practically constant at 72 h (Fig. 7a).
At 24 h, the number of viable cells harvested from the
HALig and Ag:HALig samples slightly increased with
respect to control, by 34 logs (Fig. 7a). At 48 h, the
number of viable cells embedded in the biofilm developed

123

17

Page 10 of 14

J Mater Sci: Mater Med (2015) 26:17

Fig. 7 (a) Number of S. aureus viable cells recovered from the

biofilms growing on the tested specimens after 24, 48 and 72 h,
respectively; (b) Absorbance values at 600 nm of the S. aureus
bacterial biofilm developed on the tested specimens after 24, 48 and
72 h, respectively
Fig. 6 Fluorescence microscopy images of nuclei of WJ-MSCs
grown on different substrata: pure HA (a); HALig (b); and Ag:HA
Lig (c) films. Magnification: 9200

on the HALig still showed increased values exceeding by

4.5 logs the number of VCCs obtained for the HA control,
whilst for the Ag:HALig coating was drastically lowered
(the recovered VCCs being by 20 logs less than control)
(Fig. 7a), suggesting the gradual and prolonged release of
silver ions from the organicinorganic composite coatings,
that interfere with the staphylococcal mature biofilm
development. The HALig and Ag:HALig specimens
exhibited a similar anti-biofilm activity against the 72 h
biofilms (reducing by 1.52.5 logs the VCCs as compared
to control) and supporting the hypothesis that Lig improves
the implants long-time resistance to staphylococcal colonization. However, even though a more significant
decrease of VCCs was observed as compared to control,
the increased number of VCCs developed at 72 h, as
compared to 24 and 48 h, is pleading for the role of silver
ions in the prevention of microbial adherence and for the

123

fact that silver ions are mostly released from the coating in
the first 48 h of incubation.
The density of the microbial cultures resulting from the
multiplication of 24 h biofilm embedded cells and measured at 600 nm proved to be much higher than for pure
HA coating control (Fig. 7b). Conversely, in the case of the
48 and 72 h biofilms, the microbial culture density was
decreasing in the presence of composite MAPLE coatings
compared to the control. Thus, as an effect of the Lig
introduction, the hydroxyapatite composite coating (HA
Lig) gained the ability to prevent the development of S.
aureus biofilm. When silver ions were incorporated
(Ag:HALig), the anti-biofilm efficiency slightly increased
(Fig. 7b).
The dynamics of P. aeruginosa biofilms on samples
were different. The biofilm developed on the pure HA film
control specimen had a gradual growth up to 72 h
(Fig. 8a). The assessment of the viable cells harvested from
the P. aeruginosa biofilms at 24 h (Fig. 8a) revealed no
significant change in the number of viable cells (the

J Mater Sci: Mater Med (2015) 26:17

Page 11 of 14

17

are present, and start to multiply later, when the antimicrobial substances (in the present case Ag ions and Lig)
have gone. Similar to S. aureus biofilms, the assessment of
the total P. aeruginosa biofilm development (viable and
dead cells) at 24 h, quantified by measuring the absorbance
at 600 nm (Fig. 8b), showed that HALig and Ag:HALig
specimens promoted the biofilm development, as compared
to the pure HA control coating. However, at 48 h, a drastic
decrease in the biofilm density was noticed for the composite coatings, HALig being more efficient than its Ag
containing counterpart. This is in good agreement with the
above mentioned VCC assays, substantiating the beneficial
effect of Lig on the improvement of anti-biofilm properties
of the HA coatings. At 72 h, both type of organicinorganic coatings proved equally efficient in preventing the
P. aeruginosa biofilm cells multiplication (Fig. 8b).
VCCs assays indicated that growth of C. famata peaked
at 72 h in the case of pure HA control coating (Fig. 9a).
Irrespective of the silver ions presence, the number of
viable cells recovered from the 24 and 48 h C. famata

Fig. 8 (a) Number of P. aeruginosa viable cells recovered from the

biofilms growing on the tested specimens after 24, 48 and 72 h,
respectively; (b) Absorbance values at 600 nm of the P. aeruginosa
bacterial biofilm developed on the tested specimens after 24, 48 and
72 h, respectively

quantitative difference being less than one log) developed

on the HALig and Ag:HALig specimens, as compared to
the pure HA control. Contrariwise, some interesting features were observed in the case of 48 h biofilms: both HA
Lig and Ag:HALig specimens drastically decreased the
number of recovered VCCs (by more than five logs) as
compared to the pure HA control coating. Both MAPLE
coatings showed similar bacteriostatic activity, irrespective
of the presence of Ag ions. Therefore, one can suggest that
the presence of Lig alone could induce increased antibacterial activity of an implant coating. All specimens showed
similar antimicrobial efficiency at 72 h. The increased
number of VCCs recovered at 72 h, as compared with 24
and 48 h, is pleading for the efficiency of the tested coatings to delay the biofilm development by preventing the
initial microbial adherence, but not to inhibit the formation
of the mature biofilm. These results could also be
accounted for by the selection of a persistent bacterial
population which enters in a state of metabolic latency and
stop multiplying, as long as the antimicrobial substances

Fig. 9 (a) Number of C. famata viable cells recovered from the

biofilms developed on the tested specimens after 24, 48 and 72 h,
respectively; (b) Absorbance values at 600 nm of the C. famata
fungal biofilm developed on the tested specimens after 24, 48 and
72 h, respectively

123

17

Page 12 of 14

biofilms was higher than that obtained for the pure HA

control coating, exceeding it with 25 logs. The HALig
and Ag:HALig composite coatings had a strong fungicidal
effect against the 72 h biofilms. These results could suggest
that the HALig and Ag:HALig coatings induce the
mature biofilm detachment from the respective surfaces.
One may assume that, similar to the case of bacterial
biofilms, HALig itself could exhibit a large spectrum of
antimicrobial activity.
The quantification of total C. famata biofilm by measuring the absorbance at 600 nm evidenced a clear antibiofilm effect for the two composite coatings, which acted
with similar efficiency (Fig. 9b).
Overall, the biological assays demonstrated that the
organicinorganic lignin-hydroxyapatite composite coatings synthesized by MAPLE could provide an efficient
protection against microbial biofilms, without inducing any
cytotoxicity towards tested WJ-MSCs.
The role of silver ions as a proficient agent against
various bacterial and fungal cultures has been demonstrated [15]. It comes therefore naturally to search for
alternate antimicrobial agents, which can work alone or in
synergy with renowned antimicrobials.
Following the principles of antibiotic therapy, in which
the risk of microbial resistance towards a drug is minimized by using antibiotherapy combinations, we propose a
new approach, in which two antimicrobial substances are
used to avoid the development of microbial resistance and
maximize the cumulative effect. When using two antimicrobial agents, the probability of microbial resistance is the
product of probabilities for resistance development used as
mono-therapy (when employing singular antimicrobial
agents) (P1?2 = P1 9 P2).
Our results reveal the potential of the natural biopolymer
lignin as a reliable antimicrobial agent for implant
coatings.

4 Conclusions
We report on the transfer by Matrix-Assisted Pulsed Laser
Evaporation of a large macromolecule of undefined molecular weightorganosolv lignin (Lig)embedded in a
hydroxyapatite film matrix. When silver was incorporated
into HA lattice, it yielded another composite, Ag:HALig.
The promptness and accuracy of the MAPLE technique was
demonstrated for deposition of such delicate, yet bulky
material, as suggested by EDS and proved by XPS and FTIR
results. The obtained nanocomposites were non-cytotoxic,
supporting a normal development and promoting the growth
of the adhered human mesenchymal cells. The microbiological assays showed that the coated composite secured a
prolonged release of silver ions, being protective both

123

J Mater Sci: Mater Med (2015) 26:17

against the initial phase of microbial colonization and the

mature biofilm development. The lignin addition boosted the
anti-microbial activity of HA doped with silver ions against
both bacterial and fungal biofilms. An implant surface
modified in such a manner could host osteogenic cell proliferation while shielding from bacteria and fungi, thus
facilitating a safe osteointegration of the medical device.
Acknowledgments INM, CR, NM(S), LD, AV acknowledge the
support of this work by Executive Unit for Financing Higher Education,
Research, Development and Innovation (UEFISCDI) of Romania under
the ID 304/2011 and TE82/2011 contracts. GES,ACP and MAH
acknowledge with thanks the financial support of TE 49/2011 research
grant. AJ was financed by the FP7 Nanotech FTM Grant Agreement
245916. AJ, SE, VMS and Dj.J acknowledge with thanks financing by
the Ministry of Education, Science and Technological Development,
Republic of Serbia, under contract No. III 45019. All authors thank M.
Enculescu for performing part of SEM investigations and G. Soricila
for technical assistance in microbiological testing.

References
1. Oldani C, Dominguez A. Titanium as a biomaterial for implants.
In: Fokter SK (ed) Recent Advances in Arthroplasty 2012; InTech, Rijeka. doi:10.5772/27413.
2. Juan L, Zhimin Z, Anchun M, Lei L, Jingchao Z. Deposition of
silver nanoparticles on titanium surface for antibacterial effect.
Int J Nanomed. 2010;5:2617. doi:10.2147/IJN.S8810.
3. Harris LG, Richards RG. Staphylococcus aureus adhesion to
different treated titanium surfaces. J Mater Sci. 2004;15:3114.
doi:10.1023/B:JMSM.0000021093.84680.bb.
4. Tang P, Zhang W, Wang Y, Zhang B, Wang H, Lin C, Zhang L.
Effect of superhydrophobic surface of titanium on Staphyococcus
aureus adhesion. J. Nanomater. 2011. doi:10.1155/2011/178921.
5. Hiby N, Ciofu O, Bjarnsholt T. Pseudomonas aeruginosa biofilms
in cystic fibrosis. Future Microbiol. 2010;5:166374. doi:10.2217/
fmb.10.125.
6. Tarquinio KM, Kothurkar NK, Goswami DY, Sanders RCJr, Zaritsky AL, LeVine AM. Bactericidal effects of silver plus titanium
dioxide-coated endotracheal tubes on Pseudomonas aeruginosa
and Staphylococcus aureus. Int J Nanomed. 2010;5:17783.
doi:10.2147/IJN.S8746.
7. Chandra J, Mukherjee PK, Leidich SD, Faddoul FF, Hoyer LL,
Douglas LJ, Ghannoum MA. Antifungal resistance of candidal
biofilms formed on denture acrylic in vitro. J Dent Res.
2001;80:9038. doi:10.1177/00220345010800031101.
8. Malm A, Chudzik B, Piersiak T, Gawron A. Glass surface as
potential in vitro substratum for Candida famata biofilm. Ann
Agric Environ Med. 2010;17:1158.
9. Ahariz M, Courtois P. Candida albicans biofilm on titanium:
effect of peroxidase precoating. Med Devices. 2010;3:3340.
doi:10.2147/MDER.S11724.
10. Akiba N, Hayakawa I, Keh ES, Watanabe A. Antifungal effects
of a tissue conditioner coating agent with TiO2 photocatalyst.
J Med Dent Sci 2005;52:2237.
11. Arweiler-Harbeck D, Sanders A, Held M, Jerman M, Ehrich H,
Jahnke K. Does metal coating improve the durability of silicone
voice prostheses? Acta Oto-laryngol. 2001;121:6436. doi:10.
1080/00016480121012.
12. Surmenev RA. A review of plasma-assisted methods for calcium
phosphate-based coatings fabrication. Surf Coat Technol.
2012;206:203556. doi:10.1016/j.surfcoat.2011.11.002.

J Mater Sci: Mater Med (2015) 26:17

13. Verron E, Bouler JM, Guicheux J. Controlling the biological
function of calcium phosphate bone substitutes with drugs. Acta
Biomater. 2012;8:354151. doi:10.1016/j.actbio.2012.06.022.
14. Catros S, Fricain JC, Guillotin B, Pippenger B, Bareille R, Remy
M, Lebraud E, Desbat B, Amedee J, Guillemot F. Laser-assisted
bioprinting for creating on-demand patterns of human osteoprogenitor cells and nano-hydroxyapatite. Biofabrication. 2011.
doi:10.1088/1758-5082/3/2/025001.
15. Maillard JY, Hartemann P. Silver as an antimicrobial: facts and
gaps in knowledge. Crit Rev Microbiol. 2013;39:37383. doi:10.
3109/1040841X.2012.713323.
16. Romling U, Balsalobre C. Biofilm infections, their resilience to
therapy and innovative treatment strategies. J Intern Med.
2012;272:54161. doi:10.1111/joim.12004.
17. Grainger DW, van der Mei HC, Jutte PC, van den Dungen JJ,
Schultz MJ, van der Laan BF, Zaat SA, Busscher HJ. Critical
factors in the translation of improved antimicrobial strategies for
medical implants and devices. Biomaterials. 2013;34:923743.
doi:10.1016/j.biomaterials.2013.08.043.
18. Schierholz JM, Morsczeck C, Brenner N, Konig DP, Yucel N,
Korenkov M, Neugebauer E, Rump AF, Waalenkamp G, Beuth J,
Pulverer G, Arens S. Special aspects of implant-associated infection in orthopedic surgery. From the pathophysiology to customtailored prevention strategies. Orthopade. 2004;33:397404.
doi:10.1007/s00132-004-0643-2.
19. Mijnendonckx K, Leys N, Mahillon J, Silver S, Van Houdt R.
Antimicrobial silver: uses, toxicity and potential for resistance.
Biometals. 2013;26:60921. doi:10.1007/s10534-013-9645-z.
20. Cazacu G, Capraru M, Popa VI. Advances concerning Lignin
utilization in new materials. In: Thomas S, Visak PM, Mathew AP
(eds). Advances in Natural Polymers: Composites and Nanocomposites. New York, Heidelberg: Springer; 2013. p. 255312.
21. Erakovic S, Jankovic A, Veljovic Dj, Palcevskis E, Mitric M,
Stevanovic T, Janackovic Dj, Miskovic-Stankovic V. Corrosion
stability and bioactivity in simulated body fluid of silver/
hydroxyapatite and silver/hydroxyapatite/lignin coatings on titanium obtained by electrophoretic deposition. J Phys Chem B.
2013;117:163343. doi:10.1021/jp305252a.
22. Erakovic S, Veljovic Dj, Diouf PN, Stevanovic T, Mitric M,
Janackovic Dj, Matic IZ, Juranic ZD, Miskovic-Stankovic V.
Investigation of silver impact on hydroxyapatite/lignin coatings
electrodeposited on titanium. Prog Org Coat. 2012;75:27583.
doi:10.1016/j.porgcoat.2012.07.005.
23. Santillan MJ, Quaranta NE, Boccaccini AR. Titania and titania
silver nanocomposite coatings grown by electrophoretic deposition from aqueous suspensions. Surf Coat Technol. 2010;205:
256271. doi:10.1016/j.surfcoat.2010.10.001.
24. Pique A. Deposition of polymers and biomaterials using the
Matrix-Assisted Pulsed Evaporation (MAPLE) process. In: R
Eason (ed). Pulsed Laser Deposition of Thin Films. ApplicationsLed Growth of Functional Materials. New Jersey: Wiley Interscience; 2007. p. 6384.
25. Cristescu R, Popescu C, Popescu AC, Grigorescu S, Duta L,
Mihailescu IN, Caraene G, Albulescu R, Albulescu L, Andronie
A, Stamatin I, Ionescu A, Mihaiescu D, Buruiana T, Chrisey DB.
Functionalized polyvinyl alcohol derivatives thin films for controlled drug release and targeting systems: mAPLE deposition
and morphological, chemical and in vitro characterization. Appl
Surf Sci. 2009;255:56004. doi:10.1016/j.apsusc.2008.09.047.
26. Cristescu R, Popescu C, Popescu AC, Grigorescu S, Duta L,
Mihailescu IN, Andronie A, Stamatin I, Ionescu OS, Mihaiescu
D, Buruiana T, Chrisey DB. Laser processing of polyethylene
glycol derivative and block copolymer thin films. Appl Surf Sci.
2009;255:560510. doi:10.1016/j.apsusc.2008.09.060.
27. Miroiu FM, Socol G, Visan A, Stefan N, Craciun D, Craciun V,
Dorcioman G, Mihailescu IN, Sima LE, Petrescu SM, Andronie

Page 13 of 14

28.

29.

30.

31.

32.

33.

34.
35.

36.
37.

38.

39.

40.

41.

42.

43.

44.

17

A, Stamatin I, Moga S, Ducu C. Composite biocompatible

hydroxyapatitesilk fibroin coatings for medical implants
obtained by Matrix Assisted Pulsed Laser Evaporation. Mater Sci
Eng. 2010;169:1518. doi:10.1016/j.mseb.2009.10.004.
Cristescu R, Popescu C, Socol G, Visan A, Mihailescu IN, Gittard
SD, Miller PR, Martin TN, Narayan RJ, Andronie A, Stamatin I,
Chrisey DB. Deposition of antibacterial of poly(1,3-bis-(p-carboxyphenoxy propane)-co-(sebacic anhydride)) 20:80/gentamicin
sulfate composite coatings by MAPLE. Appl Surf Sci. 2011;257:
528792. doi:10.1016/j.apsusc.2010.11.141.
Sygnatowicz M, Tiwari A. Controlled synthesis of hydroxyapatite-based coatings for biomedical application. Mater Sci Eng.
2009;29:10716. doi:10.1016/j.msec.2008.08.036.
Erakovic S, Jankovic A, Ristoscu C, Duta L, Serban N, Visan A,
Mihailescu IN, Stan GE, Socol M, Iordache O, Dumitrescu I,
Luculescu CR, Janackovic Dj, Miskovic-Stankovic V. Antifungal
activity of Ag:hydroxyapatite thin films synthesized by pulsed
laser deposition on Ti and Ti modified by TiO2 nanotubes substrates. Appl Surf Sci. 2014;293:3745. doi:10.1016/j.apsusc.
2013.12.029.
Grumezescu AM, Andronescu E, Ficai A, Bleotu C, Chifiriuc
MC. Chitin based biomaterial for antimicrobial therapy: fabrication, characterization and in vitro profile based interaction with
eukaryotic and prokaeryotic cells. Biointerface Res Appl Chem
2012; 5:43845.
Cros A. Charging effects in X-ray photoelectron spectroscopy.
J Electron Spectrosc Relat Phenom. 1992;59:114. doi:10.1016/
0368-2048(92)85008-U.
Johansson LS, Campbell JM. Reproducible XPS on biopolymers:
cellulose studies. Surf Interface Anal. 2004;36:101822. doi:10.
1002/sia.1827.
Guo JB, Tao ZY, Luo XG. Analysis of bamboo lignin with FTIR
and XPS. Acta Chim. Sin. 2005; 63:153640.
Kalaskar DM, Ulijn RV, Gough JE, Alexander MR, Scurr DJ,
Sampson WW, Eichhorn SJ. Characterisation of amino acid
modified cellulose surfaces using ToF-SIMS and XPS. Cellulose.
2010;17:74756. doi:10.1007/s10570-010-9413-y.
http://en.wikipedia.org/wiki/Lignin. Accessed 28 Jan 2014.
Markovic M, Fowler BO, Tung MS. Preparation and comprehensive characterization of a calcium hydroxyapatite reference
material. J Res Natl Inst Stand Technol. 2004;109:55368.
doi:10.6028/jres.109.042.
Guo GS, He CN, Wang ZH, Gu FB, Han DM. Synthesis of titania
and titanate nanomaterials and their application in environmental
analytical chemistry. Talanta. 2007;72:168792. doi:10.1016/j.
talanta.2007.03.039.
Kline LM, Hayes DG, Womac AR, Labbe N. Simplified determination of lignin content in hard and soft woods via UV-spectrophotometric analysis of biomass dissolved in ionic liquids.
BioResources. 2010; 5:13661383.
Rana R, Langenfeld-Heyser R, Finkeldey R, Polle A. FTIR
spectroscopy, chemical and histochemical characterisation of
wood and lignin of five tropical timber wood species of the
family of Dipterocarpaceae. Wood Sci Technol. 2010;44:22542.
doi:10.1007/s00226-009-0281-2.
Popescu CM, Popescu MC, Singurel G, Vasile C, Argyropoulos
DS, Willfor S. Spectral characterization of eucalyptus wood. Appl
Spectrosc. 2007;61:116877. doi:10.1366/000370207782597076.
Fan M, Dai D, Huang B. Fourier transform infrared spectroscopy
for natural fibres. In: S Salih, editor. Fourier TransformMaterials
Analysis. 2012, InTech; p. 4568.
Hergert HL. Infrared spectra of lignin and related compounds. II.
Conifer lignin and model compounds. J Org Chem. 1960;25:40513.
doi:10.1021/jo01073a026.
Tejado A, Pena C, Labidi J, Echeverria JM, Mondragon I.
Physico-chemical characterization of lignins from different

123

17

Page 14 of 14

sources for use in phenol-formaldehyde resin synthesis. Bioresour

Technol. 2007;98:165563. doi:10.1016/j.biortech.2006.05.042.
45. Grumezescu AM, Holban AM, Andronescu E, Mogosanu GD,
Vasile BS, Chifiriuc MC, Lazar V, Andrei E, Constantinescu A,
Maniu H. Anionic polymers and 10 nm Fe3O4@UA wound
dressings support human foetal stem cells normal development
and exhibit great antimicrobial properties. Int J Pharm. 2013.
doi:10.1016/j.ijpharm.2013.08.026.
46. Bianco P, Riminucci M, Gronthos S, Robey PG. Bone marrow
stromal stem cells: nature, biology, and potential applications.
Stem Cells. 2001;19:18092. doi:10.1634/stemcells.19-3-180.
47. Forte G, Franzese O, Pagliari S, Pagliari F, Di Francesco AM,
Cossa P, Laudisi A, Fiaccavento R, Minieri M, Bonmassar E, Di
Nardo P. Interfacing Sca-1pos mesenchymal stem cells with biocompatible scaffolds with different chemical composition and
geometry. J Biomed Biotechnol. 2009. doi:10.1155/2009/910610.

123

J Mater Sci: Mater Med (2015) 26:17

48. Zhao L, Chu PK, Zhang Y, Wu Z. Antibacterial coatings on
titanium implants. J Biomed Mater Res B. 2009;91:47080.
doi:10.1002/jbm.b.31463.
49. Schmidmaier G, Lucke M, Wildemann B, Haas NP, Raschke M.
Prophylaxis and treatment of implant-related infections by antibiotic-coated implants: a review. Injury. 2006;37:S10512. doi:10.
1016/j.injury.2006.04.016.
50. Agnihotri S, Mukherji S, Mukherji S. Immobilized silver nanoparticles enhance contact killing and show highest efficacy: elucidation of the mechanism of bactericidal action of silver.
Nanoscale. 2013;5:732840. doi:10.1039/C3NR00024A.
51. Arumugam SK, Sastry TP, Sreedhar B, Mandal AB. One step
synthesis of silver nanorods by autoreduction of aqueous silver
ions with hydroxyapatite: an inorganicinorganic hybrid nanocomposite. J Biomed Mater Res. 2007;80:3918. doi:10.1002/
jbm.a.30895.